A high-throughput combination screen identifies NT-1 as a superior compound to overcome KRAS G12D inhibitor resistance

Background: Colorectal cancer (CRC) is the second leading cause of cancer-related mortality. KRAS mutations, present in approximately 40% of CRC cases. FDA-approved inhibitors exist for KRAS G12C , but this mutation represents only a small subset of CRC, and resistance frequently develops through canonical or alternative survival pathways. KRAS G12D is the most prevalent allele in CRC; however, there are currently no FDA-approved inhibitors and few agents in advanced development. To address KRAS resistance, we developed a high-throughput combinatorial screening platform using both cell lines and patient-derived organoids (PDOs) with fully annotated molecular profiles to identify synergistic drug combinations capable of overcoming resistance. Method: To demonstrate the utility of this platform for identifying G12D inhibitor (G12Di) resistance mechanisms and synergistic partners, we performed a large-scale combinatorial drug screen in both parental and resistant KRAS G12D mutant CRC cell lines. The screening library consisted of approximately 2,600 kinase inhibitors and 3,500 FDA-approved or clinical-stage compounds, tested both as single agents and in combination with a preclinical KRAS G12D inhibitor. Viability was assessed using the ATP-based luminescence assay CellTiter-Glo. Compounds demonstrating robust synergy were prioritized for secondary validation using 8×8 dose-response matrices. The most promising compounds were validated in KRAS G12D PDOs to confirm translational potential. Result: Multiple compounds displayed synergistic activity with KRASi. Notably, "NT-1," a novel analog of the FDA-approved EGFR inhibitor osimertinib, demonstrated significant synergy (synergy score >10) at nanomolar concentrations across both sensitive and resistant CRC models. Compared with other EGFR inhibitors (osimertinib, erlotinib, afatinib), NT-1 achieved greater inhibitory effects at lower doses. These findings were extended to combinations with emerging pan-KRAS inhibitors, where NT-1 consistently outperformed approved EGFR inhibitors and cetuximab, producing deeper suppression of p-EGFR and p-ERK, and stronger induction of apoptosis. Ex vivo validation using KRAS G12D PDOs confirmed the translational potential of these combinations, demonstrating that NT-1 can mitigate both intrinsic and acquired resistance to G12Di. Ongoing in vivo studies are assessing NT-1 in combination with G12D selective and pan-KRAS inhibitors to delineate mutation-specific versus pathway-level vulnerabilities. Conclusion: Our combinatorial screening platform provides novel biological insights into KRAS G12D resistance mechanisms in CRC and effectively identifies drug combinations with immediate clinical utility. The combination of NT-1 with G12D selective or pan-KRAS inhibitors represents a promising therapeutic strategy to overcome KRAS G12D resistance.